Ablation of the Sox11 Gene Results in Clefting of theSecondary Palate Resembling the Pierre Robin Sequence*□S

Received for publication, September 8, 2015, and in revised form, January 20, 2016 Published, JBC Papers in Press, January 29, 2016, DOI 10.1074/jbc.M115.690875

Huarong Huang1, Xiaojuan Yang1, Meiling Bao, Huanhuan Cao, Xiaoping Miao, Xiaoyun Zhang, Lin Gan,Mengsheng Qiu, and Zunyi Zhang2

From the Institute of Developmental and Regenerative Biology, Zhejiang Key Laboratory for Mammalian Organogenesis andRegeneration, College of Biological and Environmental Science, Hangzhou Normal University, Zhejiang 310036, China

Mouse gene inactivation has shown that the transcription fac-tor Sox11 is required for mouse palatogenesis. However,whether Sox11 is primarily involved in the regulation of palato-genesis still remains elusive. In this study, we explored the roleof Sox11 in palatogenesis by analyzing the developmental mech-anism in cleft palate formation in mutants deficient in Sox11.Sox11 is expressed both in the developing palatal shelf and in thesurrounding structures, including the mandible. We found thatcleft palate occurs only in the mutant in which Sox11 is directlydeleted. As in the wild type, the palatal shelves in the Sox11mutant undergo outgrowth in a downward direction and exhibitpotential for fusion and elevation. However, mutant palatalshelves encounter clefting, which is associated with a malposi-tioned tongue that results in physical obstruction of palatal shelfelevation at embryonic day 14.5 (E14.5). We found that loss ofSox11 led to reduced cell proliferation in the developing man-dibular mesenchyme via Cyclin D1, leading to mandibular hyp-oplasia, which blocks tongue descent. Extensive analyses of geneexpression in Sox11 deficiency identified FGF9 as a potentialcandidate target of Sox11 in the modulation of cell proliferationboth in the mandible and the palatal shelf between E12.5 andE13.5. Finally we show, using in vitro assays, that Sox11 directlyregulates the expression of Fgf9 and that application of FGF9protein to Sox11-deficient palatal shelves restores the rate ofBrdU incorporation. Taken together, the palate defects pre-sented in the Sox11 loss mutant mimic the clefting in the PierreRobin sequence in humans.

Cleft palate and cleft lip are among the most common con-genital birth defects in humans, with a prevalence rangingbetween 1/700 and 1/1000 (1). In the mouse, palatogenesis isinitiated at embryonic day 11.5 (E11.5)3 when the maxillaryprominences outgrow as bilateral protrusions in the oral cavityto form paired palatal shelves (2). As development progresses,

the palatal shelves undergo extensive growth vertically alongboth sides of the developing tongue until E14.0, when the pal-atal shelves rapidly elevate into the horizontal position abovethe tongue. Horizontal growth continues until the bilateralshelves achieve midline contact to form a transient midline epi-thelial seam at E14.5. The degradation of this seam completesthe formation of the secondary palate around E15.0. Factorsthat interfere with growth, elevation, or fusion can result in theformation of cleft palate (2– 4).

In addition to the cleft palate caused by intrinsic factors,clefting of the secondary palate occurs as a consequence ofdefects in other craniofacial structures. Clinically, it is referredto as the Pierre Robin sequence (PRS), which is comprised ofmandibular hypoplasia (micrognathia or retrognathia), dis-placed tongue position, and cleft secondary palate (5). Previousstudies from a few mutant mouse strains, including deficienciesin Hoxa2, Snail1/2, Prdm16, Alk2, Sox9, and Erk2, have shownthat the cleft secondary palate resulting from the failed palatalelevation is a consequence of the physical obstruction by a mal-positioned tongue (6 –12), partially mimicking the clefting ofPRS in humans (2, 13, 14). In many of the murine mutant mod-els of PRS-like clefting, the expression of the gene of interest isdetected in the palatal shelves themselves in addition to therelevant structure, like the mandible (2). However, the regula-tory mechanism of how the loss of the gene of interest results inprimary defects and influences the development of the PierreRobin sequence-like palate clefting still remains unclear.

Sox11, together with Sox4 and Sox12, makes up the Sox Csubgroup of a family of transcription factors with a Sry-relatedHMG box (Sox) DNA binding domain (15, 16). The mouse andhuman genomes contain 20 orthologous pairs of Sox genes, andthese 20 genes are divided into eight subgroups on the bases ofsequence similarity and genomic organization (17). SoxC genesare all single-exon genes and encode a protein containing twohighly identical functional domains: a Sox DNA-bindingdomain (84% identity) in the N-terminal half of the protein anda transactivation domain (66% identity) at the C terminus (18,19). SoxC genes have been implicated in the widespread andhighly overlapping expression of the SoxC genes in the devel-oping mouse embryo (19 –22). In orofacial development, Sox4and Sox12 share similar expression domains, with Sox11 beingexpressed in the developing palatal shelves and tooth germ epi-thelium (19). These features suggest that the three SoxC pro-teins could exhibit similar functional properties. However,although SOX4 maps to human chromosome 6p23, a regionimplicated by several CLP linkage studies (23–26), SOX12

* This work was supported by Natural Science Foundation of China Grant31071287 and Natural Science Foundation of Zhejiang Province GrantLZ12C12002 (to Z. Z) and Natural Science Foundation of ChinaGrant 31101046 and Natural Science Foundation of Zhejiang ProvinceGrant LY15C070005 (to H. H.). The authors declare that they have no con-flicts of interest with the contents of this article.

□S This article contains supplemental Figures 1 and 2.1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Hangzhou Normal Univer-

sity, 16 Xuelin St., Xiasha, Hangzhou, Zhejiang 310036, China. Tel.: 86-571-2886-7677; E-mail: zunyi_zhang@idrbio.org.

3 The abbreviations used are: E11.5, embryonic day 11.5; PRS, Pierre Robinsequence; MEE, medial epithelial edge.

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(20p12) and SOX11 (2p25) in humans map to a region notimplicated in cleft lip/cleft palate by linkage studies. Sox4-nullmutant mice die in mid-gestation because of severe cardiacoutflow tract defects (27). Sox12-null mice have no obviousmalformations and undergo a normal lifespan and fertility (22).Sox11-null mutants exhibit neonatal lethality, with cleft palatein 100% of the mutant mice and CLP in 70% of the mutant mice(21). The mutant pups also usually have cardiac defects, pulmo-nary hypoplasia, asplenia, eyes open at birth, omphalocele, andother defects (21). Thus, the Sox11 mutant provides a candidatemodel for evaluating the etiology of SoxC genes associated withhuman cleft palate. However, little is known about the intrinsicrole of Sox11 in palatogenesis. In this study, using a mutantdeficient in mouse Sox11, we explore the potential role of Sox11 in the regulation of palatal growth by analyzing the cellularand molecular mechanisms by which the loss of Sox11 results inthe formation of cleft secondary palate. We uncover that thefailure of tongue descent in the Sox11 loss mutant physicallyhampers elevation of the palatal shelves that otherwise arecapable of elevation and fusion, resulting in the cleft secondarypalate. We also reveal that Sox11 is necessary for growth of themandible and the palatal shelves, in which Sox11 acts upstreamof Fgf9 in regulating cell proliferation.

Experimental Procedures

Animals—Generation of the Sox11fx/fx mice has beendescribed previously (28). Mouse strains for Wnt1-Cre, Dermo1-Cre, Osr2-IRESCre, K14-Cre, Foxg1-Cre, Nestin-Cre, and EIIa-Cre were purchased from The Jackson Laboratory. The morn-ing that a virginal plug was detected was considered E0.5. Thetransgenic mice were maintained in a C57/B6 background in astandard animal vivarium. All of the animal experimental pro-tocols in this study were approved by The Committee of Labo-ratory Animals (HNU-M2010-0701), Hangzhou Normal Uni-versity, Hangzhou, China.

Histology, Immunohistochemistry, and in Situ Hybrid-ization—Embryos were collected at the desired developmentalstages and fixed in 4% paraformaldehyde overnight at 4 °C. Forsectioning of in situ hybridization, embryos were dehydratedthrough graded alcohols, embedded in paraffin, and sectionedat 12 �m. In situ hybridization was carried out as describedpreviously, with digoxigenin-labeled antisense RNA probes(29). To conduct immunohistochemical staining, samples werefixed for 1 h in 4% paraformaldehyde in PBS, transferred to 30%sucrose in PBS, and stored at 4 °C overnight. Frozen sections(12 �m) were rinsed in PBS and blocked in 0.3% Triton X-100and 2% BSA in PBS for 1 h at room temperature. Sections werewashed three times in PBST (0.1% Triton X-100/PBS), blockedin 5% BSA for 30 min, and incubated with primary antibodiesdiluted with 5% BSA at 4 °C overnight in a humid chamber.Sections were subsequently washed in PBST, three times for 10min each. Secondary antibodies (1:1000) diluted in 5% BSAwere applied for 30 min in the dark. 3.3�-diaminobenzidinesubstrate kit was used for peroxidase (Vector Laboratories).The peroxidase reaction was stopped by rinsing with PBS fol-lowed by 2% paraformaldehyde fixation. Primary antibodiesused in this study were commercially purchased from Abcam:Cyclin D1 (ab134175), CyclinD2 (ab3085), Ki67 (ab15580),

Fgfr9 (ab71395), Fgfr1 (ab636010), and Fgfr2 (ab10648). Forhistology, serial sections of 7 �m were stained with hematoxy-lin and eosin. The tongue heights were measured on the basis ofserial coronal sections of embryonic heads between E13.5 andE16.5 as described previously (8). Frontal sections through pal-atal shelves were selected from the anterior, middle, and poste-rior regions, with the middle region consisting of sectionsthrough the maxillary first molar tooth buds.

Cell Proliferation and TUNEL Assays—Cell proliferationrates were measured by BrdU labeling, and apoptosis was deter-mined using the TUNEL assay. Briefly, timed pregnant micewere injected intraperitoneally with BrdU solution (5 mg/100 gof body weight) (29) from a BrdU labeling and detection kit(Roche) 20 min prior to being sacrificed. Embryonic heads (n �3 for both the wild type and mutant) were fixed in 10% neutralformalin and then processed for paraffin sectioning at 5 �m forimmunohistochemical staining according to the instructions ofthe manufacturer. BrdU-positive cells were counted (�20 con-secutive fields at �40 magnification from three samples forboth the wild type and mutants) and calculated as the percent-age of labeled cells among total nuclear stained cells within adefined arbitrary area. Statistical significance was calculatedusing Student’s t test. Apoptosis was assayed by TUNEL stain-ing using the in situ cell death assay kit according to the instruc-tions of the manufacturer (Roche).

In Vitro Organ Culture and Protein Bead Implantation—Forin vitro palatal fusion assays, secondary palatal shelves weredissected in cold PBS from E13.5 embryos. The bilateral palatalshelves were placed close to each other with the dorsal sideupward on a Nucleopor filter membrane in TROWELL organculture in a chemical defined medium as described previously(29). Forty-eight hours after culture, explants were collectedand fixed in 4% paraformaldehyde for histological staining asdescribed above.

For in vitro palatal shelf elevation assays, roller cultures wereperformed essentially as described previously (8). The embry-onic head with either the mandible or tongue removed wasplaced into a 20-ml glass culture tube containing 2 ml ofmedium. The culture tube was secured in a vertical position ona rotary apparatus rotating at a speed of 4 rpm in an incubator at37 °C and 5% CO2. Samples were harvested after 24 h of culture,and they were then processed for histological examination.

For bead implantation experiments, Affi-Gel blue-agarosebeads (Bio-Rad) were soaked in proteins as described previ-ously (29). The protein concentrations used were consistentthroughout all experiments. The beads were washed in PBS andthen incubated for 40 min at 37 °C in 100 �g/ml FGF9 (R&DSystems). Control beads were soaked with similar concentra-tions of BSA under the same conditions.

Expression Vectors and Promoter-Luciferase Constructs—The Sox11 expression vector was constructed by subcloning aSox11 cDNA fragment containing the full-length protein-cod-ing region into the pcDNA3-TOPO (Invitrogen) expressionvector. To generate the Fgf9-luciferase reporter plasmid, an1868-bp fragment (�1857 to �11 bp) containing the Sox11putative binding site was amplified from C57/B6L mousegenomic DNA and then subcloned into the pGL2-Basic vector(Promega) at the HindIII site in the multiple cloning region.

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The following primers were used for cloning the Fgf9 promoter:5�-AAG CTT AAA TTC ATT GCT TCC TCC GTC T-3� (for-ward) and 5�-AAG CTT CCA TAG CAA CGT AAG TGCCTG-3� (reverse). For the construction of the pGL2-Fgf9-Mutluciferase plasmid, the Sox11 binding site (AACAAAG mutatedtoATAGAAT)wasmutatedfromthepGL2-Fgf9 luciferasecon-struct using QuikChange XL mutagenesis (Stratagene) follow-ing the instructions of the manufacturer.

Cell Culture, Transfection, and Luciferase Reporter Assays—293T cells were cultured in DMEM supplemented with 10%fetal bovine serum and 1% penicillin/streptomycin. For lucifer-ase reporter assays, cells were plated in 24-well tissue cultureplates (Corning) and co-transfected with 0.1 �g of a luciferasereporter vector, 0.1 �g of the pRL-Renilla luciferase expressionvector (Promega), and 0.1 �g of the Sox11 expression vector.Transfections were performed using Lipofectamine 2000(Invitrogen) reagent in accordance with the instructions of themanufacturer. Luciferase activity was measured 48 h aftertransfection using the Dual-Luciferase assay kit (Promega) witha TD-20/20 luminometer (Turner Designs). Reporter expres-sion was normalized to co-transfected Renilla luciferase activ-ity, and the normalized value (RLUpGL2/RLUpRL-TK) wasused for statistical analysis. Transfection data were summa-rized from six repeated experiments.

ChIP Assay—293T cells were transiently transfected withpFLAG-Sox11-expressing vectors using Lipofectamine 2000(Invitrogen). The ChIP assay was realized with the Imprintchromatin immunoprecipitation kit (Sigma-Aldrich). For eachChIP reaction, 250,000 cells were used. The size of the soni-cated DNA fragments used for immunoprecipitation wasbetween 400 and 800 bp in length. Purified chromatin wasimmunoprecipitated using an anti-FLAG M2 antibody (Sigma).Eluted DNA fragments were purified to serve as templates forthe PCR amplification. The input fraction corresponded to 2%of the chromatin solution before immunoprecipitation. IgGcontrol served as the negative control to the FLAG antibody.

The primers used to amplify the area containing thepredicted Sox11 binding site were 5�-CTT CCC TCT CGCACG CCG TC-3� (forward) and 5�-GCG TCC CCA ACA CAAGCA CT-3� (reverse), resulting in a 173-bp fragment (P2). Theprimers used as a negative control were 5�-CGA GGCT CCGGGA CGC GGT CC-3� (forward) and 5�-ACG TTG GCG GCGGCG ACA AA-3� (reverse), resulting in a 195-bp fragment (P1).Amplification was carried out using the following conditions:one cycle at 95 °C for 2 min, followed by 37 cycles of 95 °C for30 s, 60 °C for 30 s, and 72 °C for 30 s and one final incubation at72 °C for 5 min.

Microarray and Quantitative Real-time PCR—For bothmicroarray and real-time RT-PCR, secondary palatal shelveswere dissected from E13.5 and E14.5 embryos for RNA extrac-tion using an RNA isolation kit (Ambion, RNAqueous-4RNA).The following primer pairs were used for quantitative RT-PCR:Fgfr1, 5�-GGT GAA CGG GAG TAA GAT CGG-3� (forward)and 5�-CCC CGC ATC CTC AAA GGA G-3� (reverse); Fgfr2,5�-CCT CGA TGT CGT TGA ACG GTC-3� (forward) and 5�-CAG CAT CCA TCT CCG TCA CA-3� (reverse); Fgf9, 5�-ATGGCT CCC TTA GGT GAA GTT-3� (forward) and 5�-TCCGCC TGA GAA TCC CCT TT-3� (reverse); Sox11, 5�-CGA

GCC TGT ACG ACG AAG TG-3� (forward) and 5�-AAG CTCAGG TCG AAC ATG AGG-3� (reverse); Sox4, 5�-GAC CTGCTC GAC CTG AAC C-3� (forward) and 5�-ACT CCA GCCAAT CTC CCG A-3� (reverse); and Sox12, 5�-GGA GAC GGTGGT ATC TGG G-3� (forward) and 5�-ATC ATC TCG GTAACC TCG GGG-3� (reverse).

Statistical Analysis—Data were analyzed for statistical signif-icance by Student-Newman-Keuls test using SYSTAT software(SYSTAT Inc.), with values of p � 0.05 considered to be signif-icant. All experiments were independently repeated at leastthree times.

Results

Sox11 Is Expressed in Developing Palatal Shelves and theMandible—Expression of Sox11 in the mouse palatal shelf hasbeen reported previously (19). To decipher whether Sox11 isinvolved in the regulation of palatal development, we per-formed in situ hybridization on sections of embryonic headusing a non-radioactively labeled Sox11 riboprobe to character-ize the localization of Sox11 mRNA in the palatal epitheliumand mesenchyme during palatogenesis. At E11.5, when the pal-atal shelves were initiated as outgrowths of maxillary processes,Sox11 transcripts were detected in the shelves (Fig. 1A). A sim-ilar expression pattern was maintained during the verticalgrowth period from E12.5 and E13.5, with a high level of tran-scripts in both the mesenchyme and the epithelium, includingthe medial epithelial edge (MEE) of the palatal shelves (Fig. 1, Band C). The expression of Sox11 persisted in both the mesen-chyme and the epithelium in E14.5, when the two bilateral pal-atal shelves met in the midline (Fig. 1D). Expression was down-regulated in the completely formed secondary palate at E15.5(Fig. 1E). These data showed that Sox11 was dynamicallyexpressed during the development of palatal shelves, whereextensive epithelial and mesenchymal interactions occur.

In addition, we noted that in situ hybridization simultane-ously displayed the expression of Sox11 in the other orofacialstructures, including the mandible (Fig. 1, A”–C”), which sharesa common origin with the palatal shelf (2, 13). The Sox11 tran-script was detected both in the epithelium and in the mesen-chyme throughout E11.5, E12.5, and E13.5, suggesting that theexpression of Sox11 is not in a palatal shelf-specific manner andthat Sox11 is also implicated in the development of thesestructures.

Loss of Sox11 Resulted in Cleft Palate Formation—A previousgene knockout study has shown that inactivation of Sox11 inmice resulted in cleft palate and cleft palate with cleft lip (21).To differentiate between the roles of Sox11 in the palatal epi-thelium versus the palatal mesenchyme, we generated the tis-sue-specific Sox11-null mutants using several tissue-specificCre mouse lines, including Wnt1-Cre, Dermo1-Cre, and Osr2-IRES-Cre for the mesenchyme and K14-Cre, Foxg1-Cre, andNestin-Cre for the epithelium. The conditional knockoutmouse line (Sox11fx/fx) was crossed with tissue-specific Crelines to inactivate Sox11 in either the mesenchyme or the epi-thelium. However, none of these tissue-specific Sox11 deletionmutants led to the formation of cleft palate when neonatal micewere assessed (data not shown). We attributed this to func-tional redundancy because of the highly overlapping expression

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of Sox11 with other Sox C genes in the palatal shelves (19). Wethen deleted Sox11 in both the epithelium and the mesenchymeby crossing EIIa-Cre mice with Sox11CKO mice to generateSox11EIIa-Cre mice. The deletion of Sox11 was confirmed byboth in situ hybridization and quantitative real-time PCR (sup-plemental Fig. 1). Interestingly, we found up-regulated expres-sion of Sox4 and Sox12 in Sox11EIIa-Cre (supplemental Fig. 1).

Prenatally collected Sox11EIIa-Cre mutant mice exhibitedcraniofacial malformations, including mandibular hypoplasia(Fig. 1, F and G) and cleft palate with 100% penetrance and cleftlip in 70% of the mutants, consistent with results from theSox11�/� mutants described previously (21). Scanning electronmicrographs showed complete clefting of the secondary palatein Sox11EIIa-Cre mutant embryos (data not shown). Histologicalanalysis during palatogenesis showed that the initial downwardgrowth of the palatal shelves occurred normally in Sox11EIIa-Cre

mice at E11.5, E12.5, and E13.5, during which the palatal shelvesof Sox11EIIa-Cre mice were indistinguishable in size and shapefrom those of the wild type (Fig. 2, A–L). At E14.5, the palatal

shelves in the wild-type embryos had already elevated to thehorizontal position and begun to fuse at the midline (Fig. 2,M–O), and the bilateral palatal shelves had merged to form thecomplete secondary palate at E15.5 (Fig. 2, S–U). However, thebilateral palatal shelves of Sox11EIIa-Cre mutant embryos failedto elevate and remained at the vertical position during thesestages (Fig. 2, P—R and V–X). It was also evident that the pala-tal shelves became regressed at the proximal portion inSox11EIIa-Cre mutants, resulting in the reduction in the size ofthe palatal shelves at E15.5 (Fig. 2, V–X). Occasional elevation

FIGURE 1. Sox11 expression in the developing palatal shelf and mandi-ble. A–E’, in situ hybridization on sections show the localization of mouseSox11 mRNA in the developing palatal shelf between E11.5 and E15.5 in wild-type mice. In the developing palatal shelf, expression of Sox11 in both themesenchyme and the epithelium is maintained throughout in the outgrow-ing palatal shelves (A, A’, B, B’, C, and C’) until the two palatal shelves meet inthe midline (D). The expression is down-regulated at E15.5, when the bilateralpalatal shelves are completely merged to form the complete palate (E). Allpanels shown are from the middle palate region. Note that the Sox11 tran-scripts were detected in both mesenchyme and epithelium in the mandibleat both E11.5 and E12.5 (A, A”, B, and B”) but is specifically detected in themesenchyme, where the Meckel cartilage and mandibular bone arise at E13.5(C and C”). F and G, a Sox11EIIa-Cre mouse displays severe craniofacial defects,including mandible hypoplasia (G), compared with the wild type (F). To,tongue; Mnd, mandible; Ps, palatal shelf.

FIGURE 2. Sox11EIIa-Cre mutants display cleft palate with retardation topalatal shelf elevation. A–X, H&E staining on sections of embryonic headsshows the histology of developing palatal shelves. A–L, downward out-growth of the palatal shelves shares similar features in Sox11EIIa-Cre mutantsand their wild-type littermates at E12.5 (A–C versus D–F) and E13.5 (G–I versusJ–L). Palatal shelves elevate to the horizontal position above the tongue dor-sum and contact each other in the midline at E14.5 in the wild type (M–O) butremain in a downward position in the Sox11EIIa-Cre mutant (P–R). In compari-son with a completely formed secondary palate in the wild type (S–U),Sox11EIIa-Cre mice still exhibit cleft palate with shelves in a downward positionat E15.5 (V–X). T, tongue; Ps, palatal shelf. Scale bars � 200 �m.

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of one or both sides of the palatal shelves occurred in laterstages in the Sox11EIIa-Cre mutant (supplemental Fig. 2), sug-gesting the possibility that Sox11 is not an intrinsic determinantfor the elevation of the palatal shelves. Instead, the loss of Sox11caused the retardation of the palatal shelf formation by delayingits elevation on time.

The Sox11EIIa-Cre Mutant Does Not Disrupt the Fusion Mech-anism of Palatal Shelves—Given the normal downward out-growth of palatal shelves and the retained elevation potential inthe palatal shelf of Sox11EIIa-Cre, the formation of cleft second-ary palate upon loss of Sox11 may be attributed to mechanismsinvolving fusion (2). To test whether the fusion competency ofpalatal shelves was altered in Sox11EIIa-Cre, we took the in vitroorgan culture in which the bilateral palatal shelves were closelyplaced on Nucleopore filters for 48 h. Histological examinationof cultured explants showed that complete fusion occurred intwo contacted palatal shelves in Sox11EIIa-Cre (n � 3). It wasevident that the epithelial seam disappeared (Fig. 3B) in a waycomparable with that which occurred in the wild type (n � 3)(Fig. 3A). These in vitro data suggest that fusion would occur inthe event that the two bilateral palatal shelves contacted eachother in Sox11EIIa-Cre. We further examined the expression ofmolecular markers critical for fusion fate of the palatal shelf in

Sox11EIIa-Cre in comparison with the wild type. In wild-typeembryos, MMP13 (matrix metalloproteinase 13) is exclusivelyexpressed in the MEE at E14.0, when palatal shelves are readyfor fusion on the horizontal position until the MEE is degener-ated (Fig. 3C) (30, 31). The proper expression of Jagged 2 in theepithelium is critical for the proper development of the palatalshelf (Fig. 3E), and inactivation of Jagged 2 resulted in the pre-mature and ectopic fusion of the palatal shelf and oral epithe-lium (32, 33). The TGF-�3 expression domain in the horizontalpalatal shelf in wild-type mice was mainly located in the MEE,with equal extension to the oral and nasal surfaces at E14 (Fig.3G). TGF-�3 signaling has been demonstrated to be essentialfor palate fusion (3). Similar to the wild-type controls, the tran-script of MMP13 was located in distal medial epithelium of thevertical palatal shelf in Sox11EIIa-Cre at E14 (Fig. 3D). Jagged 2was also expressed in palatal and oral epithelium inSox11EIIa-Cre, comparable with the wild type at E13.5 (Fig. 3F).The TGF-�3 mRNA was localized to the distal epithelium ofthe vertical palatal shelf at E14 in the Sox11EIIa-Cre mutant (Fig.3H). Thus, the comparable expression patterns of marker mol-ecules suggest that the fusion fate of MEE in the Sox11 mutantis not changed despite the retarded elevation at E14. This con-clusion was further supported by the positive TUNEL stainingin the medial portion of the downward palatal shelf at E14.5 inthe Sox11EIIa-Cre mutant (Fig. 3J), when apoptosis occurredin the midline seam in the wild-type control (Fig. 3I). Takentogether, these data suggest that the altered fusion program ofthe palatal shelf is not the reason for cleft palate in Sox11EIIa-Cre

mice.Malformed Tongue Position in Sox11EIIa-Cre Caused Physical

Retardation of Palatal Elevation—On the other hand,Sox11EIIa-Cre mutant mice also showed a marked reduction inMeckel cartilage size at E13.5 (Fig. 4B) and in mandibularlength and displaced tongue position during palatal elevationby E14.5 along the anterior-posterior axis, as exhibited byabnormal height in comparison with the wild-type littermates(Fig. 4, A–C), suggesting that the displaced tongue position wasattributable to physical obstruction causing the retardation ofpalatal shelf elevation (2).

To determine whether the displaced tongue positionretarded palate elevation in Sox11EIIa-Cre mice, we testedwhether the palatal shelves possessed the intrinsic ability ofelevation by in vitro organ culture on a rotary apparatus. E13.5embryonic heads with their mandible or tongue removed wereplaced in a bottle subjected to roller culture. Samples were thencollected and processed for histological examinations. Asshown in Fig. 4D, the palatal shelves in samples of Sox11EIIa-Cre

mutant (n � 6/6), similar to their wild-type littermates (n �6/6), were able to elevate after 24 h in rolling culture. Theseobservations indicate that the loss of transcription factor Sox11does not intrinsically alter the morphogenetic potential for ele-vation but rather delay it. It also suggests that the palatal shelvesin the Sox11 loss mutant still possess intrinsic competency forelevation.

Sox11 Is Required for Development of the Mandible—Theaforementioned results, together with the expression of theSox11 transcript in mandibular tissue, prompted us to examinethe potential mechanism by which ablation of Sox11 led to

FIGURE 3. Fusion of palatal shelves is not impaired in Sox11EIIa-Cre mice. Aand B, histological sections show that the palatal shelves from E13.5Sox11EIIa-Cre embryos (B), like those in the wild-type control (A), fused withcomplete degradation of the midline seam in contact in an in vitro organ.Arrows indicate the merged cells in the midline seam. C–H, in situ hybridiza-tion shows that the expression of Mmp13 (C and D), Jagged-2 (E and F), andTgf-�3 (G and H) is comparable in wild-type and Sox11EIIa-Cre mice. I and J,TUNEL assay shows cell apoptosis. Cell death is evident in the midline epithe-lial seam in the wild type (I) and in distal epithelial cells of the downwardpalatal shelf in the Sox11EIIa-Cre mutant (J). Ps, palatal shelf; T, tongue.

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growth defects of the mandibular primordia. To understand thecellular mechanism forming a hypoplastic mandible in theabsence of Sox11, we assessed the regulation of cell prolifera-tion in the mandibular mesenchyme by immunohistochemistryusing antibodies against Ki67 and CyclinD1. A decrease inCyclin D1 expression was demonstrated in the mandibularmesenchyme at E11.5 through E13.5 in Sox11EIIa-Cre (Fig. 5,A–F), suggesting that cell proliferation was affected throughregulation of a type-D Cyclin. Coincidently, the mandible

displayed a remarkable decrease in Ki67 expression in mes-enchymal cells in and around the Meckel cartilage at E13.5(Fig. 5, G–I). Taken together, these data provide evidencethat the Sox11 deletion resulted in compromised mandibulargrowth through regulation of mesenchymal cell prolifera-tion. Consistent with this suggestion, we detected that theexpression of Runx-2, an early marker for differentiation ofosteoblasts, was compromised in Sox11EIIa-Cre at E11.5 andE13.5 (Fig. 5, J–M).

FIGURE 4. Displaced tongue position obstructs palatal elevation in Sox11EIIa-Cre. A, measurement of tongue height along the anterior-posterior axis inSox11EIIa-Cre and wild-type controls. Note that tongue height at E13.5 is comparable in Sox11EIIa-Cre and wild-type controls but remained significantly increasedat E14.5, E15.5, and E16.5 in Sox11EIIa-Cre. *, p � 0.05 (n � 5); **, p � 0.01 (n � 5). A, anterior; M, middle; P, posterior. Data are represented as the mean � S.D. B,skeletal preparations show the shorter mandibles in Sox11EIIa-Cre compared with wild-type mice. Scale bars � 1 mm. C, relative measurement of mandibularlength. **, p � 0.01 (n � 5). Data are represented as the mean � S.D. D, histological staining on sections of embryonic head explants by in vitro rolling culture.The elevation of palatal shelves is achieved either by removal of the tongue or removal of the mandible. Arrows point to the comparable horizontal positionsin Sox11EIIa-Cre and wild-type controls. Ps, palatal shelf.

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Sox11 Is Required for Proper Palatal Shelf Growth—BecauseSox11 is expressed in the developing palatal shelves, to investi-gate the intrinsic role of Sox11, we also examined whether therewere alterations in cell proliferation and cell survival duringpalate development in Sox11 mutant mice. No differences incell apoptosis were found in the palatal shelves Sox11EIIa-Cre

mutant embryos at E12.5 and at E13.5 (data not shown). Cellproliferation in E12.5 palatal shelves in Sox11EIIa-Cre was alsocomparable with that in wild-type tissue by BrdU incorporationassay (data not shown). However, at E13.5, we detected a 25%reduction (p � 0.01) in the palatal mesenchyme and a 19%reduction (p � 0.01) in palatal epithelial cells in Sox11EIIa-Cre

mutant embryos (Fig. 6, B–C) in comparison with their wild-type littermates (Fig. 6, A, A’, and C). Similarly, proliferation ofboth epithelial and mesenchymal cells in the proximal bendregion of the palatal shelf was also significantly reduced inSox11EIIa-Cre mutant embryos (Fig. 6, B, B”, and D) in compar-ison with the wild-type littermates (Fig. 6, A, A”, and D).

To evaluate whether Sox11 may regulate D-type Cyclin-mod-ulated cell proliferation in palatal shelves, we examined theexpression of cyclin D1 and D2 in the palatal shelves ofSox11EIIa-Cre embryos by in situ hybridization and immunohis-tochemistry. Down-regulated expression of cyclin D1 protein(Fig. 6, E and F) and cyclin D2 mRNA in mesenchymal cells (Fig.

6, G and H) was exhibited. These data are consistent with ourdata from BrdU incorporation assays (Fig. 6, A–D).

Fgf9 Is the Downstream Target of Sox11 in Palatal and Man-dibular Growth—To explore the molecular mechanism bywhich Sox11 deletion results in cleft palate formation, we ana-lyzed gene expression profiles by microarray using RNAextracted from E13.5 and E14.5 palatal shelf tissues fromSox11EIIa-Cre and wild-type control mice (n � 2/genotype) toidentify the potential downstream genes of Sox11. In parallelcomparisons, we uncovered dozens of probe sets representingtranscripts that were differentially expressed (�1.5-fold, �5%false discovery rate (Fig. 7A). Among these genes, Fgf9 is the onedifferentially expressed (Fig. 7A) and implicated in palatogen-esis (34, 35). We confirmed the down-regulation of Fgf9 byquantitative real-time PCR using mRNA extracted from palatalshelve tissues at E13.5 and E14.5 (Fig. 7B) and in situ hybridiza-tion (Fig. 7, C and C’ versus D and D’). Interestingly, we alsofound that expression of Fgf9 was markedly reduced in the mes-enchymal site where the mandibular bone arises in E13.5Sox11EIIa-Cre compared with the wild type (Fig. 7, C” versus D”),suggesting that Fgf9 expression is a downstream target of Sox11in the mandible. Moreover, we detected a decrease in theexpression of the Fgf9 receptor Fgfr2 (36, 37) in the mandibularbone-forming site and the palatal shelf (Fig. 7, E–F”) but not

FIGURE 5. Cell proliferation in the mandibular mesenchyme is reduced in Sox11EIIa-Cre. A–H’, immunohistochemistry shows a decrease in Cyclin D1expression during mandible development in the mutant mandible at E11.5 (B versus A), E12.5 (D versus C), and E13.5 (F versus E). A significant reduction in Ki67is evident in the mesenchyme (mc) in and surrounding Meckel cartilage at E13.5 (H versus G). G’ and H’ are enlarged pictures of the boxed areas in G and H,respectively. I, relative proliferation as number of Ki67-positive cells in the boxed area in the wild type (G) versus Sox11EIIa-Cre (H). Arrows indicate the developingmandibular bone. Data are mean � S.D. of three independent samples. J—M, in situ hybridization showing the expression of Runx2 in the mandibularmesenchyme at E11.5 (J and K) and the mesenchyme surrounding Meckel cartilage at E13.5 (L and M).

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Fgfr1 (Fig. 7, G and H). These data suggest that Fgf9 and Fgfr2are potential downstream genes of Sox11 in the developingmandible and palatal shelf.

To further determine whether the transcription factor Sox11regulates developmental processes by directly binding to Fgf9,we tested the potential binding of Sox11 to the regulatoryregion of Fgf9 by in vitro ChIP assays. Sox11 protein binds to theconsensus sequence (5�-(A/T)(A/T)CAA(A/T)G-3�) in the

minor groove of DNA and can function either as a transcrip-tional activator or repressor (38, 39). By using rVista software toanalyze conserved regions within the Fgf9 promoter, we iden-tified the potential Sox11 binding site, which was located in the�1671 to �1664 bp region upstream of the transcription startsite (Fig. 8A). We utilized the oligo pairs (see “ExperimentalProcedures”) that amplify either the potential binding site ofSox11 in the regulatory regions of Fgf9 (Fig. 8A, P2) or non-binding site oligo pairs as a negative control (Fig. 8A, P1). PCRshowed that, after immunoprecipitation of linked chromatin,there was a specific enrichment of FLAG-tagged Sox11 to aDNA fragment that corresponded to a potential site with anti-bodies against FLAG compared with IgG controls (Fig. 8B).Luciferase reporter assays were further performed to test thefunctional significance of the Sox11 binding region (Fig. 8, Aand C). The reporter was significantly activated by overexpres-sion of CMV-Sox11. In contrast, these luciferase activities weredramatically diminished when the Sox11 binding site wasdeleted in the Fgf9-promoter vector (Fig. 8C). In summary, wefound that transcription factor Sox11 activates Fgf9 expressionby directly binding to this promoter region of Fgf9.

Fgf9 Maintains the Cell Proliferation of the Palatal Shelf inthe Absence of Sox11—To investigate whether Fgf9 maintainscell proliferation downstream of Sox11, we detected cell prolif-eration in the palatal shelves in organ culture by implanted Fgf9protein-saturated beads. BrdU was applied for 30 min at theend of the culture period. Immunostaining showed that theratio of BrdU incorporation was significantly increased byimplanted Fgf9 beads but not by BSA control beads (Fig. 8, Dand E), indicating that Fgf9 protein treatment restored the cellproliferation of Sox11EIIa-Cre palatal shelves.

Discussion

The formation of cleft palate with or without cleft lip result-ing from the loss of mouse Sox11 has been known for a decade(21). However, the question of whether or how Sox11 isinvolved in the regulation of mammalian palatogenesis has notbeen addressed. In this study, we found that the cleft secondarypalate in the Sox11 loss mutant is mainly due to a failed palatalshelf elevation. Interestingly, loss of Sox11 influences the pro-liferation of the mandibular mesenchymal cells, resulting in asmall mandible (hypoplasia). As a result of the reduced mandi-ble size in the Sox11 loss mutant, the tongue failed to movedownward mechanically, preventing palatal shelf elevationfrom a vertical to a horizontal position, causing cleft secondarypalate. We thus provide evidence that extrinsic influence is themajor factor in clefting of the secondary palate of the Sox11mutant, resembling the human PRS. Similar murine modelshave been revealed in the Prdm16 loss-of-function mutant andloss of mesenchymal signaling for Erk2 or Fgfr (12, 14, 40).

Morphogenetic events in murine palatogenesis, particular inthe elevation of the palatal shelves from a vertical to a horizon-tal position, depends on both intrinsic factors from the shelfitself and the extrinsic forces coinciding with the downwardmovement of the tongue (2, 13, 41). Proper cell proliferation isessential for the proper growth of craniofacial structures. InSox11EIIa-Cre mice, the mandible is evidently narrow and small,and the tongue remains heightened above the palatal shelves on

FIGURE 6. Cell proliferation of the palatal shelf is reduced in Sox11EIIa-Cre

mice. A and B, immunohistochemistry shows the BrdU incorporation in E13.5palatal shelves. In comparison with the wild type (A), the BrdU-labeled cells inthe Sox11EIIa-Cre mutant were significantly reduced in both the epithelium andthe mesenchyme (B). A’, A”, B’, and B”, large magnification of the boxed regionsrepresent the distal palatal region (A’ and B’) and bend region (A” and B”) in Aand B, respectively. Scale bars � 100 �m. C, statistical data show the relativeproliferation folds in the distal palatal shelf of the wild type (A’) andSox11EIIa-Cre mutant (B’). D, statistical analysis shows the relative proliferationfolds in the bend region in wild-type control (A”, n � 7) and Sox11EIIa-Cre

mutant mice (B”, n � 7). E and F, immunostaining shows a decrease in cyclinD1 expression in the proximal region of the palatal shelf in the Sox11EIIa-Cre

mutant (F, arrowhead) in comparison with the wild type (E, arrowhead). E’ andF’ are enlarged pictures of the boxed areas in E and F, respectively. G and H, insitu hybridization exhibits reduced expression of cyclin D2 in the proximalregion of the palatal shelf in Sox11EIIa-Cre mice (F, arrowhead) compared withthe wild-type mice (G, arrowhead). T, tongue; Ps, palatal shelf.

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both sides. On the other hand, the mutant palatal shelves with-out the tongue are able to elevate in an in vitro organ culture.These results, together with data of the enriched Sox11 tran-script in the mandibular mesenchyme and the proportionallysmall Meckel cartilage and mandibular bone in the Sox11mutant, favor a non-intrinsic palate role for Sox11 during pal-atal shelf elevation. Formation of Meckel cartilage in mousedevelopment occurs at approximately E13.5 and is closely cor-related with mandibular outgrowth (42) 1 day before palatalelevation. In the Sox11EIIa-Cre mutant, the impaired growth ofMeckel cartilage may influence mandible size and cause cleftingin the secondary palate (11, 40).

In addition, we also show that expression of Sox11 in thepalatal shelf is required for the maintenance of proper palatalgrowth. Sox11 may regulate cell proliferation in the proximalbending region on the nasal side of the palatal shelf throughD-cyclins, such as cyclin D1 and cyclin D2, suggesting a regula-tory role for Sox11 in the maintenance of cell proliferation inthe palatal mesenchyme. This indication is consistent with thefact that the oronasal patterning is highly regulated throughepithelium-mesenchyme interactions in the developing palatalshelf (43), in which the interaction between Fgf7 and Shh in thenasal region of the palatal mesenchyme could be one of the

candidate pathways to determine the oronasal patterning of thepalatal shelf in mice (43).

The expression pattern of Sox11 in the mandibular tissues isconsistent with a function in the regulation of cell proliferation.Sox-C genes encode regulatory transcription factors critical forthe cell cycle both in vitro and in vivo (18, 44 – 46). SOX4 over-expression was shown to promote cell cycle arrest and apopto-sis of HCT116 colon carcinoma cells (47). Morpholino-tar-geted disruption of Sox11 in zebrafish also identified that Sox11plays a crucial role in enhancing osteoblast differentiation byfacilitating the proliferation of mesenchymal and osteoblastprogenitor cells (45). BrdU incorporation assays have shown asignificant reduction in cell proliferation in the neural tube, thebranchial arches, and somites of Sox4/Sox11 double mutantmice (48). Highly overlapping transcript locations of Sox11 andSox12 in the mouse palatal shelf have been described previously(19). Our data reveal that the expression of Sox4 and Sox11partially overlaps in the epithelium and mesenchyme of thedeveloping palatal shelf (supplemental Fig. 1A). It is thus rea-sonable to predict the possibility that the function of Sox11 inmouse palatogenesis may be compensated for by Sox4 andSox12. The functional redundancy of SoxC genes has beendescribed in the development of the CNS (18, 19). None of the

FIGURE 7. Sox11 acts upstream of Fgf9 expression in the palatal shelf and mandible. A, heatmap of representative microarray probe sets representingtranscripts that were differentially expressed (�1.5-fold, �5% false discovery rate). Mut, Sox11EIIa-Cre. B, RT-PCR using RNA extracted from E13.5 and E14.5palatal shelves. The Sox11 transcript is completely deleted in Sox11EIIa-Cre. The expression levels of Fgf9 and Fgfr2 but not Fgfr1 are reduced in the Sox11EIIa-Cre

mutant in comparison with that in the wild type. Data are represented as the mean � S.D. and are representatives of at least three independent experiments.C–H, in situ hybridization shows decreased expression of Fgf9 (D) and Fgfr2 (F), but not Fgfr1 (H versus G), in E13.5 Sox11EIIa-Cre palatal shelves and the mandible(D) compared with the wild type (C). Boxed regions specify reduced expression of Fgf9 in the epithelium (D’ versus C’) and Fgfr2 in the mesenchyme of the bendregion of the palatal shelf (F’ versus E’). Note the markedly reduced expression of mesenchymal Fgf9 (D” versus C”) and Fgfr2 (F” versus E”) in the E13.5 mandible.Arrows indicate the palatal shelf epithelium in C’, the mandibular bone-forming site in C”, the mesenchyme in the bend region of the palatal shelf in E’, and themandibular bone-forming site in E”.

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single Sox C gene deletions in mice exhibited any CNS defects(21, 22), indicating the functional compensation for the loss ofany individual SoxC gene (18) by other SoxC genes. Recentcompound mutations of Sox C genes have also clearly shownthat the more the SoxC genes were deleted, the more severewere the defects that resulted. For instance, Sox4/Sox11 dou-ble-null mice die at early organogenesis at E10.5, indicating thatthree SoxC genes are redundantly required for successfulorganogenesis (48). However, this seems not to be the case inthe developing palatal shelf, where deletion of Sox11 results inclefting in palate and lip even though the expression of Sox4 andSox12 is up-regulated prior to the palatal shelf elevation inthe Sox11EIIa-Cre mutant. Nevertheless, the question of whetherSox11 function can be replaced by the other SoxC genes stillremains to be addressed by genetic replacement of Sox11 withother SoxC genes. Our data reveal the regulatory effect of thetranscription factor Sox11 on the cell proliferation of both mes-enchymal and epithelial cells.

Sox C transcription factors have been identified as the directregulators of several genes critical for mouse organogenesis. Inhumans, Tcfap2a (TFAP2A) maps to human chromosome6p24, a region repeatedly implicated in linkage studies ofhuman CLP (24 –26, 49). The Sox11 transcription factor has abinding site in the regulatory region of Tcfap2a, which drives

the expression of Tcfap2 in cranial neural crest cells and theirderivatives, such as the mesenchyme of the nasal prominences.Chimeric experiments where Tcfap2-null cells were mixedwith wild-type cells resulted in mouse embryos with variousisolated defects, including cleft palate, suggesting that theproper regulatory effect on Tcfap2 transcription is essential forhuman facial development (50, 51). SoxC genes bind directly tothe promoter region to promote the expression of Tead2 (48).Teads encode transcription factors that are associated withYes-associated protein (YAP) and transcriptional co-activatorwith PDZ-binding motif (TAZ) to function as co-transcriptionfactors to regulate the cell proliferation and survival underlyingthe Hippo signaling pathway (52).

Our study reveals that Sox11 acts upstream of Fgf9 in man-dibular mesenchymal cells as the mandibular bone is arising.This observation is further supported by biochemical evidencethat Sox11 directly regulates the transcription of Fgf9, whichacts in turn as a mitogen in the developing mandibular mesen-chyme and palatal shelf (35). Additionally, we show that Sox11expression in the epithelium and the mesenchyme of the devel-oping palatal shelf is present until completion of palatal devel-opment (19), which also suggests that Sox11 is involved in themolecular control of palatal growth. Sox11 has been found toregulate Fgf9 in the epithelium of the palatal shelf and receptor

FIGURE 8. Sox11 directly regulates Fgf9 expression. A, schematic of the mouse Fgf9 gene from the upstream promoter region of the transcription start site(TSS, angled arrow) to the downstream encoding region (Fgf9). A putative Sox11 binding position (A/T)(A/T)CAA(A/T)G-3�) is found in the promoter region.Primer pairs were designed to amplify a 197-bp promoter region containing a Sox11 binding site (P2) and promoter region containing no Sox11 binding site(P1). B, ChIP assays of Sox11 protein binding to endogenous Fgf9. Chromatin extracts from 293T cells transiently expressing FLAG-tagged Sox11 protein wereprecipitated in the presence of nonspecific mouse IgG or antibody against FLAG. PCR products using primer pairs amplifying P2 and control P1 from theimmunoprecipitated chromatin and input sample were resolved by agarose gel electrophoresis. C, Fgf9-luciferase reporter containing a Sox11 binding site andexpression vector of Sox11 (CMV-Sox11) were transiently transfected into 293T cells. Reporter expression was normalized to co-transfected Renilla luciferaseactivity, and the normalized value (RLUpGL2/RLUpRL-TK) was used for statistical analysis. **, p � 0.01. Data are represented as the mean � S.D. and arerepresentatives of at least three independent experiments. D and E, Fgf9 protein restores cell proliferation of the palatal shelf in Sox11EIIa-Cre. D, immunostainingshows the BrdU-labeled cells in the palatal shelves in organ culture (arrows). BrdU incorporation was reduced in Sox11EIIa-Cre in comparison with the wild type.However, the number of BrdU-positive cells was increased when Fgf9 protein beads were implanted in Sox11EIIa-Cre. E, statistical analysis of BrdU labeling inpalatal shelves implanted with either Fgf9 beads or BSA control. **, p � 0.01 (n � 5).

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Fgfr2 (36) in the nasal side mesenchyme of the palatal shelf.Fgfr-mediated control of cell proliferation has previously beenrecognized in the lift movement of palatal shelves (53). Ablationof Fgfr1 in the ectomesenchyme results in a cell proliferationdelay and cleft palate by impeding palatal shelf elevation priorto shelf fusion (53). However, in contrast to the clefting second-ary palate in our Sox11 loss mutation that highlights the extrin-sic influence of a malpositioned tongue and micrognathia onthe clefting the secondary palate, the cleft palate in loss of func-tion in Fgfr1 is due to the disturbance of cell signaling to delaycell proliferation in both the mesenchyme and epithelium ofdeveloping palatal shelves, preventing the shelves from properelevation and fusion (53). Moreover, Fgf9 associated with Pitx2has been identified to mediate TGF-� signaling and regulatecell proliferation in the palatal mesenchyme during mousepalatogenesis (35). Thus, we suggest the existence of a Sox11/Fgf9/Fgfr2 regulatory pathway in controlling the growth of themandible and palatal shelf, consistent with the critical role ofFgf9 in the regulation of growth (34).

In summary, although the palatal shelf in Sox11 mutant micefails to elevate on time, it possesses the morphogenetic poten-tial for elevation and fusion. Moreover, loss of Sox11 does notresult in the ectopic, premature fusion between the downwardpalatal epithelium and other oral epithelium that occurred inJag2�/�, Fgf10�/�, and Mn1�/� mutant mice (32, 33, 54), par-tially because of the normal Jagged-2 expression in the Sox11mutant palatal shelf. The results that facial structural impair-ments, such as mandibular dysplasia (shorter in length) andtongue displacement, accompany cleft secondary palate inSox11 mutant mice support that Sox11 deletion during palato-genesis is evidently associated with secondary effects in theSox11EIIa-Cre mutants (21), mimicking the clefting in PRS inhumans.

Author Contributions—H. H. and Z. Z. designed the research. X. Z.collected animal materials. H. H., X. Y., M. B., X. M., and H. C. per-formed the experiments. H. H. and X. Y. analyzed the data. L. G. andM. Q. helped perform the analysis with constructive discussions.Z. Z. wrote the manuscript. All authors reviewed the results andapproved the final version of the manuscript.

Acknowledgments—We thank all members of the Zhang laboratoryat the Institute of Developmental and Regenerative Biology, Hang-zhou Normal University for suggestions during the generation of thesedata.

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Sox11 Ablation Mimics the Pierre Robin Sequence

7118 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 13 • MARCH 25, 2016

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Xiaoyun Zhang, Lin Gan, Mengsheng Qiu and Zunyi ZhangHuarong Huang, Xiaojuan Yang, Meiling Bao, Huanhuan Cao, Xiaoping Miao,

the Pierre Robin Sequence Gene Results in Clefting of the Secondary Palate ResemblingSox11Ablation of the

doi: 10.1074/jbc.M115.690875 originally published online January 29, 20162016, 291:7107-7118.J. Biol. Chem. 

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